PROJECT SUMMARY: Clostridium difficile infection (CDI) is a leading nosocomial infection and the primary identifiable cause of antibiotic-associated diarrhea. It can lead to multiple CDI recurrences, sepsis, toxic megacolon, and death. Recurrent CDI complicates 20% of primary episodes of disease and is associated with an increased risk of death and a poor quality of life. While probiotics or fecal transplantation have met with some success in breaking the cycle of recurrent CDI, there remains a need for simple, safe, and cost-effective solutions to this problem. Prostaglandins (PGs) are lipid molecules involved in many aspects of health and disease. The PGs, especially the molecule PGE2, are important to gastrointestinal health and medications that block their synthesis, known as nonsteroidal anti-inflammatory drugs (NSAIDs), can cause gastrointestinal problems such as stomach ulcers. In fact, misoprostol, a PGE analogue, is FDA approved to prevent ulcers in patients taking NSAIDs. We recently applied a published and publicly available computational algorithm to perform a phenome-wide association study (PheWAS) based on ICD-9 billing code data in a disease-agnostic cohort of ~40,000 patients to identify potential novel genotype-phenotype associations related to sequence variations in the genes for the targets of misoprostol (PGE2 receptors). This analysis predicted a possible new indication for misoprostol to treat (or prevent) C. difficile colitis. Given emerging epidemiological data suggesting that NSAID use increases the risk for CDI and our new preliminary data in a mouse CDI model showing that exposure to NSAIDs worsens CDI severity while misoprostol alleviates the disease, we hypothesize that misoprostol can be repurposed to prevent recurrent C. difficile colitis. We envision a clinical study using misoprostol to prevent recurrent CDI but there are unanswered questions that must be addressed before any such clinical trial. These relate particularly to the timing and dose of the drug. We therefore propose two Aims to (1) define the optimal dose and timing of administration of oral misoprostol in a mouse model of relapsing CDI and (2) determine the correlates of protection by misoprostol in a mouse model of relapsing CDI. These studies will examine the impact of misoprostol on factors known to govern susceptibility to CDI, including changes to the gut microbiome and metabolome, tissue inflammation, and the generation of biomarkers of colitis. These studies are a critical first step in moving toward clinical studies of CDI treatment and/or prevention that exploit the beneficial effects of PGE2 on colon health. The successful completion of these studies will have immediate impact on new clinical studies of CDI and may provide evidence for more rationale use of NSAIDs or prostaglandin analogues in high CDI-risk patients.